Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation

Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation Meng-jie Zhou, Jing Wu, Liu-xiu Hu, Wen-song Hu, Jun-bao Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2351994/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jun, 2023 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 4 You are reading this latest preprint version Abstract Menaquinone-7 (MK-7), a highly valuable member of the vitamin K2 series, is an essential nutrient for humans. It plays an important role in the treatment of coagulation, osteoporosis, promotion of liver function recovery and prevention of cardiovascular diseases. In this study, in order to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of metabolic synthesis of MK-7 by mutant strain Bacillus subitilis 168 KO-SinR (BS168 KO-SinR) was analyzed by adding surfactants. The results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm, as indicated by scanning electron microscopy and flow cytometry. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. qRT-PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the results of electron microscope showed that the addition of surfactant changed the permeability of cell membrane. The research results of this paper provide a certain reference value for the industrial development of MK-7 prepared by fermentation. Bacillus subtilis Surfactants Synthetic pathway Scanning electron microscope qRT-PCR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction MK-7, a very valuable member of the fat-soluble vitamin K2 family, is a polyene compound that consists of a 2- methyl-1,4- naphthoquinone ring structure with a side chain consisting of seven isoprene units (Berenjian et al. 2015; Hu et al. 2017; Zhao et al. 2020). MK-7 is reported to be a component of the microbial plasma membrane and an important electron transport carrier in the respiratory chain (Berenjian et al. 2013; Fujimoto et al. 2012). It has the advantages of long half-life and high biological affinity in human body, thus playing an important role in the prevention of osteoporosis, cardiovascular diseases and peripheral artery diseases (Gast et al. 2009; Miyake et al. 2001; Shea and Holden 2012; Vos et al. 2012). Bacillus subtilis ( B . subtilis ), as a safe production strain for the synthesis of MK-7, is attracting extensive attention. However, due to its low synthesis efficiency and high price, it is difficult to meet the market demand. Therefore, how to improve its output has become an urgent problem to be solved. At present, the microbial synthesis of MK-7 has mainly focused on Bacillus subtilis natto ( B. subtilis natto ), Bacillus subtilis , Bacillus amyloliquefaciens and Bacillus Flavobacterium (Sato et al. 2001; Tani et al. 1986). We have learned that static fermentation of B. subtilis natto is the optimal method for the production of MK-7. Among them, AYDIN’s research group (Berenjian et al. 2013) and Pennsylvania AL1 research group (Mahdinia et al. 2018) in the United States utilized static fermentation of B. subtilis natto to produce a large number of biofilms to synthesize vitamin K2, and the formation of these biofilms significantly promoted the synthesis of vitamin K2. Therefore, the formation of B. subtilis biofilm is of great significance for promoting the anabolism of vitamin MK-7. In the previous experiment, we constructed the mutant BS168 KO-SinR by knocking out the SinR gene, a key inhibitor of biofilm regulation in B. subtilis . The synthetic ability of MK-7 was significantly improved. However, under the static condition, mutant strains synthesized a large number of biofilms which were enriched on the surface of the culture medium. As the main components of biofilms were extracellular polysaccharides, extracellular proteins and extracellular DNA (Branda et al. 2005; O'Toole et al. 2000; Parsek and Greenberg 2005), the biofilms had a certain adhesion capacity, and bacteria were accumulated in a large number of biofilms. At this time, the synthesis of MK-7 was mainly performed intracellularly. Ikeda (Ikeda and Doi 1990) reported that MK-7 was composed of two parts in a submerged fermentation system. One part forms water-soluble extracellular compounds during the fermentation process, while the other part exists in the cells as fat-soluble MK-7. However, due to its relatively large molecular weight, it is difficult to directly diffuse across the membrane. After synthesis in vivo, it is located on the electron transport chain of B. Subtilis , and only part of it is secreted outside the cell (Kurosu and Begari 2010). In addition, the continuous accumulation of intracellular vitamin K2 not only causes toxicity to cells, but also causes feedback inhibition, thus limiting the amount of product synthesis. Therefore, it is very important to increase the extracellular secretion of MK-7 against our mutant strain. By increasing the extracellular secretion of extracellular MK-7 of the mutant strain, we can stimulate the production of intracellular MK-7 and finally increase the total production of MK-7. In previous studies, surfactants have been added to cultures to increase yield, and low concentrations of surfactants have been reported to increase membrane permeability by eliminating the limitation of intracellular diffusion of target metabolites, thereby increasing extracellular productivity. For example, Fang et al. proved that in the synthesis of vitamin K2 by fermentation of Bacillus Flavobacterium , when 1% POE was added to the culture medium, the membrane permeability of Bacillus Flavobacterium was changed and VK2 could be effectively leaked from inside to outside of the cell, thereby relieving the feedback inhibition of the intracellular products and increasing the synthesis amount of VK2 to 25.55 ± 0.76 mg/L, 252.4% higher than that of the control (Fang et al. 2018). Although significant advances have been made in increasing the yields of these compounds and there are many reports of the effects of surfactants, the use of these surfactants in the synthesis of MK-7 in B. Subtilis industrial fermentation is still limited. In this work, firstly, surfactants (anionic surfactants, ionic surfactants, nonionic surfactants and zwitterionic surfactants) that enhance the synthesis of MK-7 by mutant strains were screened, and then the concentration of surfactant was optimized. Specifically, the structural changes of bacterial biofilm and cell morphology after surfactant treatment were observed by scanning electron microscopy. The permeability of cell membrane and the change of membrane potential were detected by flow cytometry. The kit was used to detect the content of NADH and NAD + and qRT-PCR was used to analyze the expression levels of genes related to biofilm formation and MK-7 synthesis. Thus, the effects of surfactants on the metabolic synthesis of MK-7 by the mutant strain was explored through these methods. Our research results will provide guidance for the industrial production of MK-7 and the comprehensive utilization of surfactants, and lay a solid foundation for the development of new health food. Materials And Methods Strains and culture method The mutant strain BS168 KO-SinR was constructed in the early stage of our laboratory and stored in our laboratory. The strain taken out from the -80 o C refrigerator was streak-cultured on LB solid medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L, and agar strip 20 g/L) to obtain a single colony. Single colonies were picked into fresh seed medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L) in a 250 ml conical flask with a working volume of 100 mL, incubated for 14-16 h at 37 o C and 200 rpm, The seed solutions were then transferred at a volume ratio of 2% to the fermentation medium (soybean peptone 50 g/L, yeast extract 20 g/L, glycerol 50 g K 2 HPO 4 3.86 g/L, KH 2 PO 4 1.62 g/L, trace elements 2 ml/L, pH 7.0) in a 250 mL conical flask with a working volume of 50 mL, and allowed to stand for fermentation for 7 days. Screening and optimization of surfactants In order to study the effects of different surfactants on the extracellular secretion of MK-7 metabolites of mutant strains and screen the optimal surfactant, 0.5% surfactants, including cationic surfactant CTAB, anionic surfactant SDS, nonionic surfactants Tween-80, PEG-200, and zwitterionic surfactants diammonium citrate and betaine, were added into the fermentation medium at first. Subsequently, the optimum concentration of surfactant was optimized. Detection of biomass and MK-7 yield Biomass measurement: The bacterial strains were fermented under different culture conditions for 7 d, and the bacterial solutions were all sucked out and centrifuged to remove the culture medium, which was then washed for 2-3 times with PBS buffer solution to completely remove the culture medium. Then centrifuge and weigh the wet weight, which is the biomass (g/L). Determination of VK2 yield: According to the reference (Fang et al. 2018), the bacterial solutions fermented for 7 d under different conditions were mixed evenly, and 2 mL of the fermentation solution was taken into a centrifuge tube, and the mixture of isopropanol and n-hexane (1:2, by volume) with four times its volume was added, and mixed evenly with a vortex mixing oscillator. After standing in the dark for 30 min, the upper extract was centrifuged, placed in a 5 mL centrifuge tube, and filtered through a 0.22 μm organic filter membrane to obtain a sample, which was measured by HPLC. The measured values were substituted into the standard curve to calculate the vitamin K2 content. Scanning electronic microscope (SEM) observation After fermentation for 7 d, the cells were centrifuged, washed twice with 0.1 M phosphate buffer and dried naturally. The cells were then fixed with 2.5% glutaraldehyde at 4 o C for 6-12 h and washed three times, 20 min each time, with 0.1 M phosphate buffer. Gradient ethanol dehydration (30%, 50%, 70%, 80%, 95% and 100% ethanol once, 20 min each time) was performed. The cells were replaced with 100% acetone (4 o C, 20 minutes each time, wash twice). The cells obtained were freeze-dried, metal sprayed and observed under a scanning electron microscope. Flow Cytometry to detect the permeability of cell membrane Fluorescent dye PI (Sigma Aldrich, St Louis, Mo., USA) was used for single staining of the mutant strain cells treated with surfactant. The bacterial solution cultured in logarithmic growth phase was centrifuged at 4 o C, 6160g for 3 min, washed with 0.2 M PBS buffer for 3 times, and resuspended in the buffer (the bacterial density was 10 7 -10 8 CFU/mL). Then, add PI dye solution with the concentration of 10 ug/mL, incubate for 15 min at 4 o C in the dark, pass through a 200-mesh nylon sieve, and place it in flow cytometry. The detection recorded at a low rate of 10,000 cells with excitation light at 488 nm was used to detect the number of positive bacteria stained with PI. Determination of NADH and NAD + (Wang et al. 2019) Mutant strains and NADH and NAD + levels treated with 0.7% Tween-80 were tested according to the kit's instructions (cominbio, China) and the method used was according to the manufacturer's instructions. The NADH and NAD + levels are quantified by colorimetric determination at 570 nm. Validation of gene expression levels for biofilm formation and MK-7 synthesis by qRT-PCR The real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the gene transcription level of the engineering strain. First of all, fresh bacteria cultured in different media were collected from the fermentation broth respectively for RNA extraction. RNA extraction and reverse transcription into cDNA: The extraction of total RNA was performed using the procedures described in the RNAiso Plus reagent (Takara). RNA concentration was determined using a nucleic acid quantitator and RNA integrity was verified by 1.5% agarose gel electrophoresis. Qualified samples were used for cDNA synthesis using the reverse transcription kit PrimeScript™ RT reagent kit with gDNA Eraser (Takara). TB green premix Ex Taq (Takara) was used for real-time quantitative PCR. CcpA gene was used as internal reference gene, and primers were designed by NCBI online website. The obtained primers were sent to Jinweizhi Biotechnology Co., Ltd. for synthesis, and the expression differences of related genes among different samples were analyzed by 2 -ΔΔCT method (Pfaffl 2001). Data Analysis All experiments were repeated three times, and the results were taken as the average value. All data were statistical and analyzed by SPSS and Origin. Results Screening and optimization of surfactants Surfactants synthesized by altering the permeability of cell membranes and increasing the target product during fermentation have been reported. For example, Liu et al. (Lu et al. 2021) reported that the addition of the surfactant Triton X-100 to the submerged fermentation of monascus could increase the cell membrane permeability and effectively promote the secretion and production of secondary metabolites. Due to the diverse types of surfactants leading to different effects, in this study, we first conducted a preliminary screening of different types of surfactants, and the results are shown in Fig. 1. Among different types of surfactants, cationic surfactants and anion surfactant showed strong toxicity to the mutant strains. When 0.5% CTAB or 0.5% SDS was added into the medium, the bacterial cells lost their activity and their growth was completely inhibited (Fig. 1a). After seven days of culture, no MK-7 was detected in the fermentation broth and the biomass was only 0.02 and 0.17 g/mL (Fig. 1c). The main reason for this kind of phenomenon is that the anionic surfactant can enter the inside of the bacteria, has a strong penetration to the cell membrane, can make the cell membrane disintegration, and membrane protein hydrophobic part combined with and separated from the membrane, can also destroy the ionic bonds and covalent bonds in protein, affect the normal growth of cells, and cationic surfactant CTAB long-chain alkyl can Pierce the cell membrane, lead to cell death. The addition of both nonionic and zwitterionic surfactants could improve the biomass of the strain and the extracellular secretion of MK-7, and the addition of different surfactants also led to different changes in the surface morphology of bacterial biofilm. Among them, the addition of zwitterionic betaine and diammonium citrate had a certain effect on the biomass of bacteria and the increase of MK-7, and made the surface of the biofilm have more wrinkles. As zwitterionic surfactants, betaine and diammonium citrate have two ionic states in the solution. The molecules of betaine and diammonium citrate have both positive and negative charges. It is possible that betaine and diammonium citrate have the adsorption capacity of non-polar solid surface monolayer, and enhance the adhesion ability of mutant strain to medium. Thus, betaine and diammonium citrate have certain influence on promoting the formation of mutant strain biofilm. Furthermore, the formation of bacterial biomass, the synthesis of MK-7 and the morphology of biofilm were changed to a certain extent. However, the addition of nonionic surfactants PEG-200 and Tween-80 exhibited stronger biofilm formation and MK-7 synthesis capabilities, and also caused the biofilm surface to present similar granular corrugated substances to gather together. In our previous studies, we also found that the formation of wrinkles could promote the connection of channel networks in the membrane and reduce the resistance of liquid flow, so that the pressure in the pipeline was lower than the atmospheric pressure outside. Such a pressure gradient could drive and promote the flow of nutrients through the biofilm (Wu et al. 2021) and finally affect the synthesis of the secondary metabolite MK-7. As a nonionic surfactant, PEG-200 does not ionize and generate ions in aqueous solution, possessing the characteristics of wetting and emulsifying. Previous studies have shown that the hydrophobic groups of nonionic surfactants can produce large repulsive volume and anti-osmotic pressure, which can affect the formation of high polymers during the formation of biofilm, described by Edgar (2010). And low concentration of PEG-200 can provide a wet environment suitable for the growth of biofilm, thus improving the biomass of the strain and secretion of MK-7 and changing the surface morphology of biofilm. In this experiment, Tween-80 showed the most significant effect on increasing the extracellular secretion of MK-7 (P < 0.05) (Fig. 1b). Specifically, the extracellular secretion of MK-7 was increased 2.3 times as compared with that of the control group. As Tween-80 showed outstanding effect on the extracellular secretion of mutant MK-7, we further optimized the concentration of Tween-80, and determined that when 0.7% Tween-80 was added, the extracellular secretion of MK-7 reached 28.8 ± 0.6 mg/L (Figure 1d), which was 2.7 times that of the control group. We can conclude that appropriate surfactants can increase biomass accumulation and target product yield during fermentation. Effects of surfactants on cell membrane morphology of BS168 KO-SinR The cell morphology of strain BS168 KO-SinR after the addition of 0.7% Tween-80 was observed by scanning electron microscopy. As shown in Fig. 2a, the cell morphology of the strain without the surfactant Tween-80 was complete, showing a typical long rod shape, and the shape was clear and full. The cell wall of BS168 KO-SinR and the surface of the cell membrane were smooth, complete and without defect marks. However, after the cells were treated with 0.7% Tween-80, significant morphological changes were observed (Fig. 2b). The cell surface was relatively rough and porous, with a large number of spherical surface-active micelles attached. We speculate that it may be due to the mutual dissolution of phospholipid bilayer and surfactant molecule and the formation of some mixed micelles, which have changed the structure of the cell membrane. There were many irregular morphological changes on the cell surface of BS168 KO-SinR, which were more conducive to the intracellular entry of substrates in the fermentation system and the secretion of product MK-7 to the outside of the cell. Effects of surfactants on cell membrane permeability of BS168 KO-SinR Propidium iodide (PI) is a positively charged nucleic acid-binding probe widely used for the evaluation of cell membrane. PI can enter dead cells to bind to DNA and RNA, but cannot enter cells with complete cell membrane structure. Further study (Hewitt and Nebe-Von-Caron 2001) found that cells stained with PI dye were not all dead cells, and cells with changed membrane permeability or damaged cell membrane could also be stained with fluorescence and detected. Thus, the effect of surfactants on the membrane permeability of the BS168 KO-SinR cell membrane was examined by PI in combination with loss of cells. Unstained cells were set as the negative control (Fig. 3a), stained cells killed at 100 o C was set as the positive control (Fig. 3b), and the area where the dead or undamaged cells were located was designated as the gate, and the cells in the gate were the bacterial cells with altered membrane permeability. The more proportion of cells in the gate indicates that the more cells with damaged cell membrane. As shown in Fig. 3, the fluorescence intensity of the mutant strain treated with Tween-80 was 48% higher than that of the control cells, and the results showed that 0.7% Tween-80 significantly improved the cell membrane permeability. The increase of cell membrane permeability may be due to the changes in the composition and content of unsaturated fatty acids in the cell membrane, which is conducive to the absorption of nutrients and the secretion of extracellular polymeric substances (Sheng et al. 2013; Zhang and Cheung 2011). This is consistent with the SEM verification results. Effects of surfactants on electron transport of mutant strains Biooxidation in mitochondria depends on the action of a series of enzymes or coenzymes on its inner membrane. These enzymes or coenzymes are arranged in a certain order on their inner membrane, forming an electron or hydrogen transport system called the electron transport chain, also known as the respiratory chain. In B. subtilis , MK-7 plays an important role in the electron transport process. In a complete electron transport chain, NADH dehydrogenase provides electrons for the electron transport chain. The electrons are transported through MK-7 and cytochrome c, and finally oxygen acts as an electron receptor to form water (Fig. 4a). Through the above experiments, we know that the addition of 0.7% Tween-80 can change the permeability of cell membrane and increase the extracellular secretion of MK-7. Therefore, in order to determine the effects of the surfactant Tween-80 on electron transfer and respiration in the cell membrane of the strain, NADH and NAD + were measured after seven days of culture. It can be seen from Fig. 4b that the level of NADH in the sample added with surfactant was higher than that in the control group, while the level of NADH + was lower than that in the control group. High NADH level can provide stable electrons, and enhanced electron transfer is conducive to the synthesis of MK-7. This is consistent with the previous study, the concentration of MK-7 in the culture system was positively correlated with the total level of NAD (Wang et al. 2019). Enhancement of the strength of the electron transport chain can increase the yield of MK-7, while the electron transport chain occurs on the cell membrane. The stability of the electron transport chain depends on the state or composition of the cell membrane. The higher the oxygen transfer efficiency in the fermentation system was, the higher the concentration of MK-7. Therefore, the addition of surfactants may enhance the strength of the electron transport chain, thereby increasing the yield of MK-7. Effects of surfactants on gene expression levels of the MK-7 synthetic pathway in BS168 KO-SinR strain To reveal the reason why the addition of surfactant enhanced the synthesis of MK-7, qRT-PCR was performed in the control group and the experimental group to determine the effect of surfactant on the gene expression level of MK-7 synthetic pathway. By comparing the expression levels of related genes in four classical modules of MK-7 synthetic pathway in the control group and the experimental group (with the addition of surfactant), as shown in Fig. 5, the biosynthesis pathway of MK-7 in B. subtilis is divided into four modules. They were the glycerol metabolism pathway (module 1), methyl erythritol phosphate (MEP) pathway (module 2), shikimic acid (SA) pathway (module 3) and MK-7 pathway (module 4). Berenjian (Berenjian et al. 2011) and Luo (Luo et al. 2016) showed that glycerol was the optimal carbon source for the production of MK-7 by fermentation of B. subtilis natto . There was only one glycerol catabolization pathway in B. subtilis , and glycerol produced dihydroxyacetone phosphate through glycerol kinase GlpK and glycerol-3-phosphate dehydrogenase GlpD, respectively, and then entered glycolytic pathway. Precursors of the SA pathway and the MEP pathway were derived from glycolytic pathway. In the glycerol metabolic pathway, the expressions of GlpF, GlpK and GlpD were up-regulated by 13.54-fold, 22.4-fold and 5.73-fold, respectively. These results indicated that the addition of 0.7% Tween-80 could increase the consumption of substrate glycerol, which was beneficial to the synthesis of glycerol-3-phosphate dehydrogenase and allowed glyceraldehyde-3-phosphate to flow into the MEP pathway and SA pathway as much as possible, thus providing a precursor for the subsequent pathway. Module II MEP pathway is the synthetic pathway of MK-7 isoprene side chain and the ratelimiting pathway of MK-7 biosynthesis. Among them, 1-deoxyxylulose-5-phosphate synthase ( dxs ) and 1- deoxyxylulose -5- phosphate reductase ( dxr ) are considered to be the rate-limiting enzymes in this pathway. The gene expression of MEP pathway is shown in Fig. 5a. In the experimental group, the gene expression levels of rate-limiting enzymes dxs and dxr were significantly increased. This result is consistent with a previous study, which found that overexpression of rate-limiting enzymes Dxs, Dxr, Idi, and MenA in B. subtilis and different combinations finally increased the MK-7 content of the recombinant strain from 4.5 mg/L to 50 mg/L (Ma et al. 2019). In addition, in this pathway, the isomerase YpqA, which catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylenephenyl diphosphate (DMAPP), was up-regulated by 25.93 times, and the pentadienyl pyrophosphate synthase HepT/HepS, which catalyzes the conversion of farnesyl pyrophosphate FPP to heptapentenyl pyrophosphate HDP, was up-regulated by 31.53 and 18.21 times, all of which favor the accumulation of isoprene side chains. The SA pathway of module III provides a precursor branch acid (CHA) for the main chain of MK-7, which is essential for cellular metabolism and serves as a precursor for the biosynthesis of three aromatic amino acids Tyrosine (Tyr ), Phenylalanine (Phe ), and tryptophan (Trp ) (Krämer et al. 2003). In the shikimic acid pathway, the expression levels of SA dehydrogenase AroD and EPSP synthetase AroD were down-regulated by 0.83 times and 1.33 times, respectively. In B. subtilis , AroD and AroE are not only useful for the synthesis of MK-7 but also precursors of aromatic amino acids. Therefore, the increased expression of these genes promotes the synthesis of aromatic amino acids, which leads to the accumulation of intracellular prephenic acid, thus exerting feedback inhibition on the SA pathway. In this pathway, SA kinase AroK and AroF were obviously up-regulated by 12.09 times and 15.11 times, respectively. It has been reported that SA kinase AroK is the key rate-limiting step of branching acid pathway. In that study, a strong promoter P43 was used to overexpress shikimic acid kinase coding gene aroK, and a recombinant strain was obtained. After six days of fermentation, the results showed that the yield of MK-7 was about 2.1 times that of the original strain B. subtilis 168 (Cui et al. 2019). The above results indicated that the branched acid was an important factor limiting the efficient synthesis of MK-7. Module IV is the last pathway used for the synthesis of MK-7. In this MK-7 synthesis pathway, the expression level of isocitrate synthase MenF was up-regulated by 28.02 times, which led to the more conversion of branched acid salt (CHA) to isobranched acid synthase (ICHA). The changes in gene expression levels mentioned above directly promoted the accumulation of important raw materials for the four pathways in the vitamin K2 synthesis pathway, namely, the glycerol cleavage pathway, the shikimic acid pathway, the methyl erythritol phosphate pathway, and the MK-7 pathway, thereby facilitating the biosynthesis of vitamin K2. Discussion Previous studies have found that biofilm formation is beneficial for MK-7 synthesis in B. subtilis (Cui et al. 2020). Therefore, we knocked out sinR gene, the key inhibitor of biofilm regulation, and got a mutant strain with more advantages in MK-7 synthesis. We revealed that the extracellular secretion level of MK-7 may be closely related to the permeability of cell membrane. When the cell structure changes, it is more beneficial for the substrate to enter the cell and the product MK-7 to be secreted out of the cell. As far as we know, this study clarified for the first time the relationship between the addition of Tween-80 and biosynthesis, cell membrane structure and electron transfer chain system of Bacillus subtilis MK-7. This work also provides new information for further increasing the output of MK-7, which is crucial for the industrial application of MK-7. As shown in Fig. 1b, among several surfactants used in the experiment, the addition of Tween -80 significantly improved the synthesis ability and extracellular secretion of MK-7. This may be because Tween-80, as one of the most important nonionic surfactants, can reduce surface and interfacial tension, improve the permeability of cell membrane to increase the content of extracellular polymeric substances, promote the adhesion of biofilm, and improve the solubility and bioavailability (Singh et al. 2007), previous studies have reported that Tween-80 is an effective stimulant in some bacteria (Zhibo et al. 2006). The size of the cells may be increased to increase the absorption efficiency of the substrate and the rate of metabolite excretion. In addition, a previous study also found that Tween-80 could also serve as an oxygen carrier to improve the oxygen transfer efficiency and promote the production of metabolites (Tu et al. 2015). In order to study whether the cell membrane has changed, the membrane permeability was examined by flow cytometry. As shown in Fig. 3, cells with damaged cell membrane were detected by nucleic acid binding probe. According to the fluorescence intensity display results, it was shown that 0.7% Tween-80 significantly improved the cell membrane permeability, which was consistent with the results verified by scanning electron microscope. Therefore, the change of cell membrane permeability may be one of the factors affecting the increase of MK-7 production of Bacillus subtilis . As we all know, bacterial cell membranes are rich in enzymes, which perform many important metabolic functions. Intracellular biological oxidation depends on electron transfer chain, and enhancing electron transfer is more beneficial to MK-7 synthesis. The stability of electron transport chain depends on the state or composition of cell membrane, and the addition of surfactant changes the structure of cell membrane, thus enhancing the strength of electron transport chain. Wang et al. found that the higher the oxygen transfer efficiency of fermentation system, the higher the concentration of MK-7 (Wang et al. 2019). In our research, we found that the MK-7 production of engineering bacteria added with 0.7% Tween-80 increased by 80.3%. This may be because relatively high concentration of MK-7 can promote the electron transfer of complex I, thus further promoting the secretion of the product to the outside of the cell. As shown in Fig 4, the electron transfer process of Bacillus subtilis is enhanced, and more MK-7 is secreted out of the cell. Finally, in order to reveal the reason of the increase of MK-7 synthesis by adding surfactants, we used real-time fluorescence quantitative PCR (qRT-PCR) to determine the effect of surfactants on the gene expression level of MK-7 synthesis pathway. According to the qRT-PCR data, most of the intermediate regulatory genes in MK-7 synthesis pathway were up-regulated in different degrees in the medium supplemented with Tween-80, which made the related genes in the whole metabolic pathway cooperate with each other and accumulated the target products. Conclusions This study showed that the addition of surfactant could indeed improve the ability of B. subtilis to produce MK-7. Under the conditions of optimizing the surfactant, it was found that the addition of 0.7% Tween-80 could significantly increase the yield of MK-7 and the total synthesis of MK-7 by 80.3%. In addition, according to the measurement of cell membrane permeability, cell morphology, transcription levels of intracellular NADH, NAD + and key genes in the MK-7 synthesis pathway, the increase in MK-7 yield in the presence of 0.7% Tween-80 was attributed to the increase in intracellular NADH level. At the same time, this study proved that the improvement of key gene expression level and the enhancement of cell membrane permeability in MK-7 synthesis pathway are beneficial to the exchange of intracellular substances and the accumulation of extracellular MK-7. These results not only provide insights into the mechanism of increasing the yield of MK-7 in the presence of surfactants, but also provide a feasible way for the efficient production of vitamin K2. Declarations Author information Corresponding Author Yan Liu− College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China; Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, 241000, Wuhu, China; orcid.org/0000-0003-0454-1181; Email: [email protected] Authors Meng-jie Zhou − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Jing Wu − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Liu-xiu Hu − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China; Anhui Zhang Hengchun Pharmaceutical Co., LTD, 241000, Wuhu, China Wen-song Hu − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Jun-bao Huang − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Xi-lin Huang − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Xu-li Gao − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Ya-ni Luo − College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China Zheng-lian Xue - College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China; Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, 241000, Wuhu, China. Acknowledgements The study was supported by the National Nature Science Foundation of China (No. 31871781，31772081), Science and technology project of Wuhu (2020yf62), the National Undergraduate Innovation and Entrepreneurship Program (No. 201910363042) and Anhui Provincial Undergraduate Innovation and Entrepreneurship Program (No. S202010363255). Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mengjie Zhou, Jing Wu, Liuxiu Hu, Wensong Hu, Junbao Huang, Xilin Huang, Xuli Gao, Yani Luo, Zhenglian Xue and Yan Liu. The first draft of the manuscript was written by Mengjie Zhou, Jing Wu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Declarations All authors have approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no relevant financial or nonfinancial interests to disclose. References Berenjian A, Chan NL, Mahanama R, Talbot A, Regtop H, Kavanagh J, Dehghani, F (2013) Effect of biofilm formation by Bacillus subtilis natto on menaquinone-7 biosynthesis. Mol Biotechnol 54(2):371–378. https://doi.org/10.1007/s12033-012-9576-x Berenjian A, Mahanama R, Kavanagh J, Dehghani F (2015) Vitamin K series: current status and future prospects. Crit Rev Biotechnol 35(2):199-208. https://doi.org/10.3109/07388551.2013.832142 Berenjian A, Mahanama R, Talbot A, Biffin R, Regtop H, Valtchev P, Kavanagh J, Dehghani, F (2011) Efficient media for high menaquinone-7 production: response surface methodology approach. 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Appl Microbiol Biotechnol 103(18):7519–7535. https://doi.org/10.1007/s00253-019-10044-5 Wu J, Li W, Zhao SG, Qian SH, Wang Z, Zhou MJ, Hu WS, WangJ, Hu LX, LiuY, Xue, ZL (2021) Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis . Microb Cell Fact 20(1):113. Zhang BB, Cheung PC (2011) A mechanistic study of the enhancing effect of Tween 80 on the mycelial growth and exopolysaccharide production by Pleurotus tuber-regium . Bioresour Technol 102(17):8323–8326. https://doi.org/10.1016/j.biortech.2011.06.021 Zhao CL, Wan YP, Tang GX, Jin Q, Zhang HL, Xu ZN (2020) Comparison of different fermentation processes for the vitamin K2 (Menaquinone-7) production by a novel Bacillus velezensis ND strain. Process Biochem 102(9):33-41. https://doi.org/10.1016/j.procbio.2020.11.029 Zhang ZB, Zeng GM, Shi JG, Liu J, Yang, WC (2006) Effect of Tween 80 and rhamnolipid on the production of protease from Pseudomonas Aeruginosa and Bacillus Subtilis . Acta Scientiae Circumstantiae 26:1152–1158. https://doi.org/10.3321/j.issn:0253-2468.2006.07.018 Luo MM, Ren LJ, Chen SL, Ji XJ, Huang H (2016) Effect of media components and morphology of Bacillus subtilis natto on menaquinone-7 synthesis in submerged fermentation. Biotechnol Bioproc E 21(6):777-786. https://doi.org/10.1007/s12257-016-0202-9 Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Published Journal Publication published 09 Jun, 2023 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Major revision 09 Dec, 2022 Editor assigned by journal 07 Dec, 2022 Submission checks completed at journal 07 Dec, 2022 First submitted to journal 06 Dec, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2351994","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":158110679,"identity":"18430c44-8e4f-4cf8-b26b-8caa55a97f8f","order_by":0,"name":"Meng-jie Zhou","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng-jie","middleName":"","lastName":"Zhou","suffix":""},{"id":158110680,"identity":"38b55997-7fd3-41ac-b541-27e85e633612","order_by":1,"name":"Jing Wu","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wu","suffix":""},{"id":158110681,"identity":"810dc336-7761-4a51-80f3-8ea6b2fa1f68","order_by":2,"name":"Liu-xiu Hu","email":"","orcid":"","institution":"Anhui Zhang Hengchun Pharmaceutical Co., LTD","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liu-xiu","middleName":"","lastName":"Hu","suffix":""},{"id":158110682,"identity":"d867e7d4-f7b3-4549-aa2d-937fff97e0be","order_by":3,"name":"Wen-song Hu","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen-song","middleName":"","lastName":"Hu","suffix":""},{"id":158110683,"identity":"bfae59c1-5676-4ada-b6ea-118e84be2ce3","order_by":4,"name":"Jun-bao Huang","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun-bao","middleName":"","lastName":"Huang","suffix":""},{"id":158110684,"identity":"17fbc419-87fc-4d64-ab2e-90a9de17ad38","order_by":5,"name":"Xi-lin Huang","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xi-lin","middleName":"","lastName":"Huang","suffix":""},{"id":158110685,"identity":"71de4cb6-d799-444a-b782-f5bfbcf45882","order_by":6,"name":"Xu-li Gao","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu-li","middleName":"","lastName":"Gao","suffix":""},{"id":158110687,"identity":"023120ef-748e-4361-9ffd-49f53da55af7","order_by":7,"name":"Ya-ni Luo","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ya-ni","middleName":"","lastName":"Luo","suffix":""},{"id":158110689,"identity":"95fe7cf4-f0a6-4309-b0cd-baf10753d746","order_by":8,"name":"Zheng-lian Xue","email":"","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zheng-lian","middleName":"","lastName":"Xue","suffix":""},{"id":158110691,"identity":"50f555a2-8b21-4fc2-8801-8d42a632815a","order_by":9,"name":"Yan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACPmYwZcNgAKbZiNDCBtGSRooWCHWYFC3sPGYSP3eclzeX7jFg+FB2mIF/dgMhh/GYSfaeuW24c84ZA8YZ5w4zSNw5QFiLBG/bbcYNN3IMmHnbgC6USCDClr9t5+zBWv4Sq0Wat+1AIlgLI3Fa2IqtZduSkzfcOVZwsOdcOo/EDQJa+PkPb7z5ts3OdsPt5o0PfpRZy/HPIKAFCFgkwBSQPACkeAiqBwLmDzAto2AUjIJRMAqwAgDVqzxRx075jwAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui Polytechnic University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2022-12-07 03:14:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2351994/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2351994/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-023-03671-8","type":"published","date":"2023-06-09T21:06:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":30124437,"identity":"47b510be-430a-4158-98d1-738fc47b2fff","added_by":"auto","created_at":"2022-12-09 15:39:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1297987,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of surfactants on mutant strains. (A) Effects of Different Surfactants on Biofilm Morphology; (B) Effect of different surfactants MK-7 synthesis amount; (C) Effects of Different Surfactants on Biomass of Mutant Strains; (D) Optimization of Tween-80 concentration\u003c/p\u003e\n\u003cp\u003eAttention：Asterisks indicate the level of statistical significance (*p \u0026lt; 0.05; **p \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/ad530e20e11cefe8859792a8.png"},{"id":30123227,"identity":"10875c53-b426-4efa-bb83-f1944a43b1c5","added_by":"auto","created_at":"2022-12-09 15:31:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":423259,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) observation of strain BS168 KO-SinR treated with different surfactants. (A): without surfactant; (B): with 0.7% Tween-80\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/c65f0812d966c0d7a79abdbd.png"},{"id":30123228,"identity":"a657d4c4-3f97-47c5-bb8b-d623dd56b836","added_by":"auto","created_at":"2022-12-09 15:31:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51781,"visible":true,"origin":"","legend":"\u003cp\u003eFCM fluorescence histogram of mutant strain treated with surfactant by PI single staining. (A) negative control; (B) positive control; (C) Untreated cell; (D) 0.7% Tween-80 treated cell\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/c47f11b2c8fe3e4b8b88d48e.png"},{"id":30123225,"identity":"dbb8c83c-19be-4bcc-a5a2-0fa48d46d4fb","added_by":"auto","created_at":"2022-12-09 15:31:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87902,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic diagram of electron transfer chain of \u003cem\u003eBacillus subtilis\u003c/em\u003e; (B) Changes of intracellular NADH and NAD\u003csup\u003e+\u003c/sup\u003e levels after adding surfactants\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/8223a59632ed56afeb092bc7.png"},{"id":30123226,"identity":"ae2b834c-df92-4b9a-bd9d-bb1617f62bdd","added_by":"auto","created_at":"2022-12-09 15:31:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169931,"visible":true,"origin":"","legend":"\u003cp\u003e(A) and (B) Transcriptional expression level of MK-7 pathway related genes; (C) The biosynthesis pathway of MK-7 in \u003cem\u003eB.subitilis\u003c/em\u003e168\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/f46ac15842f8b31a874cef68.png"},{"id":44732543,"identity":"1c4951f0-09b4-45cf-bed2-89470f39aa35","added_by":"auto","created_at":"2023-10-16 21:55:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1239666,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/51abd259-7e90-4f1a-bca4-95093e89c724.pdf"},{"id":30123224,"identity":"d1f784c4-a6a8-42b6-bc58-72118a2a86ab","added_by":"auto","created_at":"2022-12-09 15:31:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31245,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2351994/v1/c3c7c2738d43833ad8d49461.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMK-7, a very valuable member of the fat-soluble vitamin K2 family, is a polyene compound that consists of a 2- methyl-1,4- naphthoquinone ring structure with a side chain consisting of seven isoprene units\u0026nbsp;(Berenjian et al. 2015; Hu et al. 2017; Zhao et al. 2020). MK-7 is reported to be a component of the microbial plasma membrane and an important electron transport carrier in the respiratory chain\u0026nbsp;(Berenjian et al. 2013;\u0026nbsp;\u0026nbsp;Fujimoto\u0026nbsp;et al. 2012). It has the advantages of long half-life and high biological affinity in human body, thus playing an important role in the prevention of osteoporosis, cardiovascular diseases and peripheral artery diseases\u0026nbsp;(Gast et al. 2009; Miyake et al. 2001; Shea and Holden 2012; Vos et al. 2012). \u003cem\u003eBacillus subtilis\u0026nbsp;\u003c/em\u003e(\u003cem\u003eB\u003c/em\u003e. \u003cem\u003esubtilis\u003c/em\u003e), as a safe production strain for the synthesis of MK-7, is attracting extensive attention. However, due to its low synthesis efficiency and high price, it is difficult to meet the market demand. Therefore, how to improve its output has become an urgent problem to be solved.\u003c/p\u003e\n\u003cp\u003eAt present, the microbial synthesis of MK-7 has mainly focused on \u003cem\u003eBacillus subtilis natto\u0026nbsp;\u003c/em\u003e(\u003cem\u003eB. subtilis natto\u003c/em\u003e), \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003eFlavobacterium\u003c/em\u003e (Sato et al. 2001; Tani et al. 1986). We have learned that static fermentation of \u003cem\u003eB. subtilis natto\u003c/em\u003e is the optimal method for the production of MK-7. Among them, AYDIN\u0026rsquo;s research group\u0026nbsp;(Berenjian et al. 2013)\u0026nbsp;and Pennsylvania AL1 research group \u0026nbsp;(Mahdinia et al. 2018)\u0026nbsp;in the United States utilized static fermentation of \u003cem\u003eB. subtilis natto\u003c/em\u003e to produce a large number of biofilms to synthesize vitamin K2, and the formation of these biofilms significantly promoted the synthesis of vitamin K2. Therefore, the formation of \u003cem\u003eB. subtilis\u003c/em\u003e biofilm is of great significance for promoting the anabolism of vitamin MK-7.\u003c/p\u003e\n\u003cp\u003eIn the previous experiment, we constructed the mutant BS168 KO-SinR by knocking out the SinR gene, a key inhibitor of biofilm regulation in \u003cem\u003eB. subtilis\u003c/em\u003e. The synthetic ability of MK-7 was significantly improved. However, under the static condition, mutant strains synthesized a large number of biofilms which were enriched on the surface of the culture medium. As the main components of biofilms were extracellular polysaccharides, extracellular proteins and extracellular DNA\u0026nbsp;(Branda et al. 2005; O\u0026apos;Toole et al. 2000; Parsek and Greenberg 2005), the biofilms had a certain adhesion capacity, and bacteria were accumulated in a large number of biofilms. At this time, the synthesis of MK-7 was mainly performed intracellularly. Ikeda\u0026nbsp;(Ikeda and Doi 1990)\u0026nbsp;reported that MK-7 was composed of two parts in a submerged fermentation system. One part forms water-soluble extracellular compounds during the fermentation process, while the other part exists in the cells as fat-soluble MK-7. However, due to its relatively large molecular weight, it is difficult to directly diffuse across the membrane. After synthesis in vivo, it is located on the electron transport chain of \u003cem\u003eB. Subtilis\u003c/em\u003e, and only part of it is secreted outside the cell\u0026nbsp;(Kurosu and Begari 2010). In addition, the continuous accumulation of intracellular vitamin K2 not only causes toxicity to cells, but also causes feedback inhibition, thus limiting the amount of product synthesis. Therefore, it is very important to increase the extracellular secretion of MK-7 against our mutant strain. By increasing the extracellular secretion of extracellular MK-7 of the mutant strain, we can stimulate the production of intracellular MK-7 and finally increase the total production of MK-7.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn previous studies, surfactants have been added to cultures to increase yield, and low concentrations of surfactants have been reported to increase membrane permeability by eliminating the limitation of intracellular diffusion of target metabolites, thereby increasing extracellular productivity. For example, Fang et al. proved that in the synthesis of vitamin K2 by fermentation of \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003eFlavobacterium\u003c/em\u003e, when 1% POE was added to the culture medium, the membrane permeability of \u003cem\u003eBacillus\u003c/em\u003e \u003cem\u003eFlavobacterium\u003c/em\u003e was changed and VK2 could be effectively leaked from inside to outside of the cell, thereby relieving the feedback inhibition of the intracellular products and increasing the synthesis amount of VK2 to 25.55 \u0026plusmn;\u0026nbsp;0.76 mg/L, 252.4% higher than that of the control\u0026nbsp;(Fang et al. 2018). Although significant advances have been made in increasing the yields of these compounds and there are many reports of the effects of surfactants, the use of these surfactants in the synthesis of MK-7 in \u003cem\u003eB. Subtilis\u003c/em\u003e industrial fermentation is still limited.\u003c/p\u003e\n\u003cp\u003eIn this work, firstly, surfactants (anionic surfactants, ionic surfactants, nonionic surfactants and zwitterionic surfactants) that enhance the synthesis of MK-7 by mutant strains were screened, and then the concentration of surfactant was optimized. Specifically, the structural changes of bacterial biofilm and cell morphology after surfactant treatment were observed by scanning electron microscopy. The permeability of cell membrane and the change of membrane potential were detected by flow cytometry. The kit was used to detect the content of NADH and NAD\u003csup\u003e+\u003c/sup\u003e and qRT-PCR was used to analyze the expression levels of genes related to biofilm formation and MK-7 synthesis. Thus, the effects of surfactants on the metabolic synthesis of MK-7 by the mutant strain was explored through these methods. Our research results will provide guidance for the industrial production of MK-7 and the comprehensive utilization of surfactants, and lay a solid foundation for the development of new health food.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and culture method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mutant strain BS168 KO-SinR was constructed in the early stage of our laboratory and stored in our laboratory. The strain taken out from the -80\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC\u0026nbsp;refrigerator was streak-cultured on LB solid medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L, and agar strip 20 g/L) to obtain a single colony. Single colonies were picked into fresh seed medium (tryptone 10 g/L, NaCl 10 g/L, yeast extract 5 g/L) in a 250 ml conical flask with a working volume of 100 mL, incubated for 14-16 h at 37\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC\u0026nbsp;and 200 rpm, The seed solutions were then transferred at a volume ratio of 2% to the fermentation medium (soybean peptone 50 g/L, yeast extract 20 g/L, glycerol 50 g K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e3.86 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.62 g/L, trace elements 2 ml/L, pH 7.0) in a 250 mL conical flask with a working volume of 50 mL, and allowed to stand for fermentation for 7 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening and optimization of surfactants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to study the effects of different surfactants on the extracellular secretion of MK-7 metabolites of mutant strains and screen the optimal surfactant, 0.5% surfactants, including cationic surfactant CTAB, anionic surfactant SDS, nonionic surfactants Tween-80, PEG-200, and zwitterionic surfactants diammonium citrate and betaine, were added into the fermentation medium at first. Subsequently, the optimum concentration of surfactant was optimized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of biomass and MK-7 yield\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiomass measurement: The bacterial strains were fermented under different culture conditions for 7 d, and the bacterial solutions were all sucked out and centrifuged to remove the culture medium, which was then washed for 2-3 times with PBS buffer solution to completely remove the culture medium. Then centrifuge and weigh the wet weight, which is the biomass (g/L).\u003c/p\u003e\n\u003cp\u003eDetermination of VK2 yield: According to the reference\u0026nbsp;(Fang et al. 2018), the bacterial solutions fermented for 7 d under different conditions were mixed evenly, and 2 mL of the fermentation solution was taken into a centrifuge tube, and the mixture of isopropanol and n-hexane (1:2, by volume) with four times its volume was added, and mixed evenly with a vortex mixing oscillator. After standing in the dark for 30 min, the upper extract was centrifuged, placed in a 5 mL centrifuge tube, and filtered through a 0.22 \u0026mu;m organic filter membrane to obtain a sample, which was measured by HPLC. The measured values were substituted into the standard curve to calculate the vitamin K2 content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electronic microscope (SEM) observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter fermentation for 7 d, the cells were centrifuged, washed twice with 0.1 M phosphate buffer and dried naturally. The cells were then fixed with 2.5% glutaraldehyde at 4\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC\u0026nbsp;for 6-12 h and washed three times, 20 min each time, with 0.1 M phosphate buffer. Gradient ethanol dehydration (30%, 50%, 70%, 80%, 95% and 100% ethanol once, 20 min each time) was performed. The cells were replaced with 100% acetone (4\u0026nbsp;\u003csup\u003eo\u003c/sup\u003eC, 20 minutes each time, wash twice). The cells obtained were freeze-dried, metal sprayed and observed under a scanning electron microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry to detect the permeability of cell membrane\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescent dye PI (Sigma Aldrich, St Louis, Mo., USA) was used for single staining of the mutant strain cells treated with surfactant. The bacterial solution cultured in logarithmic growth phase was centrifuged at 4 \u003csup\u003eo\u003c/sup\u003eC, 6160g for 3 min, washed with 0.2 M PBS buffer for 3 times, and resuspended in the buffer (the bacterial density was 10\u003csup\u003e7\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e CFU/mL). Then, add PI dye solution with the concentration of 10 ug/mL, incubate for 15 min at 4 \u003csup\u003eo\u003c/sup\u003eC in the dark, pass through a 200-mesh nylon sieve, and place it in flow cytometry. The detection recorded at a low rate of 10,000 cells with excitation light at 488 nm was used to detect the number of positive bacteria stained with PI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of NADH and NAD\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003csup\u003e(Wang et al. 2019)\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMutant strains and NADH and NAD\u003csup\u003e+\u003c/sup\u003e levels treated with 0.7% Tween-80 were tested according to the kit\u0026apos;s instructions (cominbio, China) and the method used was according to the manufacturer\u0026apos;s instructions. The NADH and NAD\u003csup\u003e+\u003c/sup\u003e levels are quantified by colorimetric determination at 570 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of gene expression levels for biofilm formation and MK-7 synthesis by qRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the gene transcription level of the engineering strain. First of all, fresh bacteria cultured in different media were collected from the fermentation broth respectively for RNA extraction. RNA extraction and reverse transcription into cDNA: The extraction of total RNA was performed using the procedures described in the RNAiso Plus reagent (Takara). RNA concentration was determined using a nucleic acid quantitator and RNA integrity was verified by 1.5% agarose gel electrophoresis. Qualified samples were used for cDNA synthesis using the reverse transcription kit PrimeScript\u0026trade;\u0026nbsp;RT reagent kit with gDNA Eraser (Takara). TB green premix Ex Taq (Takara) was used for real-time quantitative PCR. CcpA gene was used as internal reference gene, and primers were designed by NCBI online website. The obtained primers were sent to Jinweizhi Biotechnology Co., Ltd. for synthesis, and the expression differences of related genes among different samples were analyzed by 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u0026nbsp;\u003c/sup\u003emethod \u0026nbsp;(Pfaffl 2001).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were repeated three times, and the results were taken as the average value. All data were statistical and analyzed by SPSS and Origin.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eScreening and optimization of surfactants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurfactants synthesized by altering the permeability of cell membranes and increasing the target product during fermentation have been reported. For example, Liu et al.\u0026nbsp;(Lu et al. 2021)\u0026nbsp;reported that the addition of the surfactant Triton X-100 to the submerged fermentation of monascus could increase the cell membrane permeability and effectively promote the secretion and production of secondary metabolites. Due to the diverse types of surfactants leading to different effects, in this study, we first conducted a preliminary screening of different types of surfactants, and the results are shown in Fig. 1. Among different types of surfactants, cationic surfactants and anion surfactant showed strong toxicity to the mutant strains. When 0.5% CTAB or 0.5% SDS was added into the medium, the bacterial cells lost their activity and their growth was completely inhibited (Fig. 1a). After seven days of culture, no MK-7 was detected in the fermentation broth and the biomass was only 0.02 and 0.17 g/mL (Fig. 1c). The main reason for this kind of phenomenon is that the anionic surfactant can enter the inside of the bacteria, has a strong penetration to the cell membrane, can make the cell membrane disintegration, and membrane protein hydrophobic part combined with and separated from the membrane, can also destroy the ionic bonds and covalent bonds in protein, affect the normal growth of cells, and cationic surfactant CTAB long-chain alkyl can Pierce the cell membrane, lead to cell death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe addition of both nonionic and zwitterionic surfactants could improve the biomass of the strain and the extracellular secretion of MK-7, and the addition of different surfactants also led to different changes in the surface morphology of bacterial biofilm. Among them, the addition of zwitterionic betaine and diammonium citrate had a certain effect on the biomass of bacteria and the increase of MK-7, and made the surface of the biofilm have more wrinkles. As zwitterionic surfactants, betaine and diammonium citrate have two ionic states in the solution. The molecules of betaine and diammonium citrate have both positive and negative charges. It is possible that betaine and diammonium citrate have the adsorption capacity of non-polar solid surface monolayer, and enhance the adhesion ability of mutant strain to medium. Thus, betaine and diammonium citrate have certain influence on promoting the formation of mutant strain biofilm. Furthermore, the formation of bacterial biomass, the synthesis of MK-7 and the morphology of biofilm were changed to a certain extent. However, the addition of nonionic surfactants PEG-200 and Tween-80 exhibited stronger biofilm formation and MK-7 synthesis capabilities, and also caused the biofilm surface to present similar granular corrugated substances to gather together.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our previous studies, we also found that the formation of wrinkles could promote the connection of channel networks in the membrane and reduce the resistance of liquid flow, so that the pressure in the pipeline was lower than the atmospheric pressure outside. Such a pressure gradient could drive and promote the flow of nutrients through the biofilm\u0026nbsp;(Wu et al. 2021)\u0026nbsp;and finally affect the synthesis of the secondary metabolite MK-7. As a nonionic surfactant, PEG-200 does not ionize and generate ions in aqueous solution, possessing the characteristics of wetting and emulsifying. Previous studies have shown that the hydrophobic groups of nonionic surfactants can produce large repulsive volume and anti-osmotic pressure, which can affect the formation of high polymers during the formation of biofilm, described by\u0026nbsp;Edgar (2010). And low concentration of PEG-200 can provide a wet environment suitable for the growth of biofilm, thus improving the biomass of the strain and secretion of MK-7 and changing the surface morphology of biofilm. In this experiment, Tween-80 showed the most significant effect on increasing the extracellular secretion of MK-7 (P \u0026lt; 0.05) (Fig. 1b). Specifically, the extracellular secretion of MK-7 was increased 2.3 times as compared with that of the control group. As Tween-80 showed outstanding effect on the extracellular secretion of mutant MK-7, we further optimized the concentration of Tween-80, and determined that when 0.7% Tween-80 was added, the extracellular secretion of MK-7 reached 28.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6 mg/L (Figure 1d), which was 2.7 times that of the control group. We can conclude that appropriate surfactants can increase biomass accumulation and target product yield during fermentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of surfactants on cell membrane morphology of BS168 KO-SinR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell morphology of strain BS168 KO-SinR after the addition of 0.7% Tween-80 was observed by scanning electron microscopy. As shown in Fig. 2a, the cell morphology of the strain without the surfactant Tween-80 was complete, showing a typical long rod shape, and the shape was clear and full. The cell wall of BS168 KO-SinR\u0026nbsp;and the surface of the cell membrane were smooth, complete and without defect marks. However, after the cells were treated with 0.7% Tween-80, significant morphological changes were observed (Fig. 2b). The cell surface was relatively rough and porous, with a large number of spherical surface-active micelles attached. We speculate that it may be due to the mutual dissolution of phospholipid bilayer and surfactant molecule and the formation of some mixed micelles, which have changed the structure of the cell membrane. There were many irregular morphological changes on the cell surface of BS168 KO-SinR, which were more conducive to the intracellular entry of substrates in the fermentation system and the secretion of product MK-7 to the outside of the cell.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of surfactants on cell membrane permeability of BS168 KO-SinR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePropidium iodide (PI) is a positively charged nucleic acid-binding probe widely used for the evaluation of cell membrane. PI can enter dead cells to bind to DNA and RNA, but cannot enter cells with complete cell membrane structure. Further study\u0026nbsp;(Hewitt and Nebe-Von-Caron 2001)\u0026nbsp;found that cells stained with PI dye were not all dead cells, and cells with changed membrane permeability or damaged cell membrane could also be stained with fluorescence and detected. Thus, the effect of surfactants on the membrane permeability of the BS168 KO-SinR cell membrane was examined by PI in combination with loss of cells. Unstained cells were set as the negative control (Fig. 3a), stained cells killed at 100 \u003csup\u003eo\u003c/sup\u003eC was set as the positive control (Fig. 3b), and the area where the dead or undamaged cells were located was designated as the gate, and the cells in the gate were the bacterial cells with altered membrane permeability. The more proportion of cells in the gate indicates that the more cells with damaged cell membrane. As shown in Fig. 3, the fluorescence intensity of the mutant strain treated with Tween-80 was 48% higher than that of the control cells, and the results showed that 0.7% Tween-80 significantly improved the cell membrane permeability. The increase of cell membrane permeability may be due to the changes in the composition and content of unsaturated fatty acids in the cell membrane, which is conducive to the absorption of nutrients and the secretion of extracellular polymeric substances\u0026nbsp;(Sheng et al. 2013; Zhang and Cheung 2011). This is consistent with the SEM verification results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of surfactants on electron transport of mutant strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiooxidation in mitochondria depends on the action of a series of enzymes or coenzymes on its inner membrane. These enzymes or coenzymes are arranged in a certain order on their inner membrane, forming an electron or hydrogen transport system called the electron transport chain, also known as the respiratory chain. In \u003cem\u003eB. subtilis\u003c/em\u003e, MK-7 plays an important role in the electron transport process. In a complete electron transport chain, NADH dehydrogenase provides electrons for the electron transport chain. The electrons are transported through MK-7 and cytochrome c, and finally oxygen acts as an electron receptor to form water (Fig. 4a). Through the above experiments, we know that the addition of 0.7% Tween-80 can change the permeability of cell membrane and increase the extracellular secretion of MK-7. Therefore, in order to determine the effects of the surfactant Tween-80 on electron transfer and respiration in the cell membrane of the strain, NADH and NAD\u003csup\u003e+\u003c/sup\u003e were measured after seven days of culture. It can be seen from Fig. 4b that the level of NADH in the sample added with surfactant was higher than that in the control group, while the level of NADH\u003csup\u003e+\u003c/sup\u003e was lower than that in the control group. High NADH level can provide stable electrons, and enhanced electron transfer is conducive to the synthesis of MK-7. This is consistent with the previous study, the concentration of MK-7 in the culture system was positively correlated with the total level of NAD\u0026nbsp;(Wang et al. 2019). Enhancement of the strength of the electron transport chain can increase the yield of MK-7, while the electron transport chain occurs on the cell membrane. The stability of the electron transport chain depends on the state or composition of the cell membrane. The higher the oxygen transfer efficiency in the fermentation system was, the higher the concentration of MK-7. Therefore, the addition of surfactants may enhance the strength of the electron transport chain, thereby increasing the yield of MK-7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of surfactants on gene expression levels of the MK-7 synthetic pathway in BS168 KO-SinR strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal the reason why the addition of surfactant enhanced the synthesis of MK-7, qRT-PCR was performed in the control group and the experimental group to determine the effect of surfactant on the gene expression level of MK-7 synthetic pathway. By comparing the expression levels of related genes in four classical modules of MK-7 synthetic pathway in the control group and the experimental group (with the addition of surfactant), as shown in Fig. 5, the biosynthesis pathway of MK-7 in \u003cem\u003eB. subtilis\u003c/em\u003e is divided into four modules. They were the glycerol metabolism pathway (module 1), methyl erythritol phosphate (MEP) pathway (module 2), shikimic acid (SA) pathway (module 3) and MK-7 pathway (module 4). Berenjian\u0026nbsp;(Berenjian et al. 2011)\u0026nbsp;and Luo\u0026nbsp;(Luo et al. 2016)\u0026nbsp;showed that glycerol was the optimal carbon source for the production of MK-7 by fermentation of \u003cem\u003eB. subtilis natto\u003c/em\u003e. There was only one glycerol catabolization pathway in \u003cem\u003eB. subtilis\u003c/em\u003e, and glycerol produced dihydroxyacetone phosphate through glycerol kinase GlpK and glycerol-3-phosphate dehydrogenase GlpD, respectively, and then entered glycolytic pathway. Precursors of the SA pathway and the MEP pathway were derived from glycolytic pathway. In the glycerol metabolic pathway, the expressions of GlpF, GlpK and GlpD were up-regulated by 13.54-fold, 22.4-fold and 5.73-fold, respectively. These results indicated that the addition of 0.7% Tween-80 could increase the consumption of substrate glycerol, which was beneficial to the synthesis of glycerol-3-phosphate dehydrogenase and allowed glyceraldehyde-3-phosphate to flow into the MEP pathway and SA pathway as much as possible, thus providing a precursor for the subsequent pathway.\u003c/p\u003e\n\u003cp\u003eModule II MEP pathway is the synthetic pathway of MK-7 isoprene side chain and the ratelimiting pathway of MK-7 biosynthesis. Among them, 1-deoxyxylulose-5-phosphate synthase (\u003cem\u003edxs\u003c/em\u003e) and 1- deoxyxylulose -5- phosphate reductase (\u003cem\u003edxr\u003c/em\u003e) are considered to be the rate-limiting enzymes in this pathway. The gene expression of MEP pathway is shown in Fig. 5a. In the experimental group, the gene expression levels of rate-limiting enzymes \u003cem\u003edxs\u003c/em\u003e and \u003cem\u003edxr\u003c/em\u003e were significantly increased. This result is consistent with a previous study, which found that overexpression of rate-limiting enzymes Dxs, Dxr, Idi, and MenA in \u003cem\u003eB. subtilis\u003c/em\u003e and different combinations finally increased the MK-7 content of the recombinant strain from 4.5 mg/L to 50 mg/L\u0026nbsp;(Ma et al. 2019). In addition, in this pathway, the isomerase YpqA, which catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylenephenyl diphosphate (DMAPP), was up-regulated by 25.93 times, and the pentadienyl pyrophosphate synthase HepT/HepS, which catalyzes the conversion of farnesyl pyrophosphate FPP to heptapentenyl pyrophosphate HDP, was up-regulated by 31.53 and 18.21 times, all of which favor the accumulation of isoprene side chains.\u003c/p\u003e\n\u003cp\u003eThe SA pathway of module III provides a precursor branch acid (CHA) for the main chain of MK-7, which is essential for cellular metabolism and serves as a precursor for the biosynthesis of three aromatic amino acids Tyrosine (Tyr ), Phenylalanine (Phe ), and tryptophan (Trp )\u0026nbsp;(Kr\u0026auml;mer et al. 2003). In the shikimic acid pathway, the expression levels of SA dehydrogenase AroD and EPSP synthetase AroD were down-regulated by 0.83 times and 1.33 times, respectively. In \u003cem\u003eB. subtilis\u003c/em\u003e, AroD and AroE are not only useful for the synthesis of MK-7 but also precursors of aromatic amino acids. Therefore, the increased expression of these genes promotes the synthesis of aromatic amino acids, which leads to the accumulation of intracellular prephenic acid, thus exerting feedback inhibition on the SA pathway. In this pathway, SA kinase AroK and AroF were obviously up-regulated by 12.09 times and 15.11 times, respectively. It has been reported that SA kinase AroK is the key rate-limiting step of branching acid pathway. In that study, a strong promoter P43 was used to overexpress shikimic acid kinase coding gene aroK, and a recombinant strain was obtained. After six days of fermentation, the results showed that the yield of MK-7 was about 2.1 times that of the original strain \u003cem\u003eB. subtilis\u003c/em\u003e 168\u0026nbsp;(Cui et al. 2019). The above results indicated that the branched acid was an important factor limiting the efficient synthesis of MK-7.\u003c/p\u003e\n\u003cp\u003eModule IV is the last pathway used for the synthesis of MK-7. In this MK-7 synthesis pathway, the expression level of isocitrate synthase MenF was up-regulated by 28.02 times, which led to the more conversion of branched acid salt (CHA) to isobranched acid synthase (ICHA). The changes in gene expression levels mentioned above directly promoted the accumulation of important raw materials for the four pathways in the vitamin K2 synthesis pathway, namely, the glycerol cleavage pathway, the shikimic acid pathway, the methyl erythritol phosphate pathway, and the MK-7 pathway, thereby facilitating the biosynthesis of vitamin K2.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have found that biofilm formation is beneficial for MK-7 synthesis in \u003cem\u003eB. subtilis\u0026nbsp;\u003c/em\u003e(Cui et al. 2020). Therefore, we knocked out \u003cem\u003esinR\u003c/em\u003e gene, the key inhibitor of biofilm regulation, and got a mutant strain with more advantages in MK-7 synthesis. We revealed that the extracellular secretion level of MK-7 may be closely related to the permeability of cell membrane. When the cell structure changes, it is more beneficial for the substrate to enter the cell and the product MK-7 to be secreted out of the cell. As far as we know, this study clarified for the first time the relationship between the addition of Tween-80 and biosynthesis, cell membrane structure and electron transfer chain system of \u003cem\u003eBacillus subtilis\u003c/em\u003e MK-7. This work also provides new information for further increasing the output of MK-7, which is crucial for the industrial application of MK-7.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 1b, among several surfactants used in the experiment, the addition of Tween -80 significantly improved the synthesis ability and extracellular secretion of MK-7. This may be because Tween-80, as one of the most important nonionic surfactants, can reduce surface and interfacial tension, improve the permeability of cell membrane to increase the content of extracellular polymeric substances, promote the adhesion of biofilm, and improve the solubility and bioavailability\u0026nbsp;(Singh et al. 2007), previous studies have reported that Tween-80 is an effective stimulant in some bacteria\u0026nbsp;(Zhibo et al. 2006). The size of the cells may be increased to increase the absorption efficiency of the substrate and the rate of metabolite excretion. In addition, a previous study also found that Tween-80 could also serve as an oxygen carrier to improve the oxygen transfer efficiency and promote the production of metabolites\u0026nbsp;(Tu et al. 2015).\u003c/p\u003e\n\u003cp\u003eIn order to study whether the cell membrane has changed, the membrane permeability was examined by flow cytometry. As shown in Fig. 3, cells with damaged cell membrane were detected by nucleic acid binding probe. According to the fluorescence intensity display results, it was shown that 0.7% Tween-80 significantly improved the cell membrane permeability, which was consistent with the results verified by scanning electron microscope. Therefore, the change of cell membrane permeability may be one of the factors affecting the increase of MK-7 production of \u003cem\u003eBacillus subtilis\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs we all know, bacterial cell membranes are rich in enzymes, which perform many important metabolic functions. Intracellular biological oxidation depends on electron transfer chain, and enhancing electron transfer is more beneficial to MK-7 synthesis. The stability of electron transport chain depends on the state or composition of cell membrane, and the addition of surfactant changes the structure of cell membrane, thus enhancing the strength of electron transport chain. Wang et al. found that the higher the oxygen transfer efficiency of fermentation system, the higher the concentration of MK-7 (Wang et al. 2019). In our research, we found that the MK-7 production of engineering bacteria added with 0.7% Tween-80 increased by 80.3%. This may be because relatively high concentration of MK-7 can promote the electron transfer of complex I, thus further promoting the secretion of the product to the outside of the cell. As shown in Fig 4, the electron transfer process of \u003cem\u003eBacillus subtilis\u003c/em\u003e is enhanced, and more MK-7 is secreted out of the cell.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, in order to reveal the reason of the increase of MK-7 synthesis by adding surfactants, we used real-time fluorescence quantitative PCR (qRT-PCR) to determine the effect of surfactants on the gene expression level of MK-7 synthesis pathway. According to the qRT-PCR data, most of the intermediate regulatory genes in MK-7 synthesis pathway were up-regulated in different degrees in the medium supplemented with Tween-80, which made the related genes in the whole metabolic pathway cooperate with each other and accumulated the target products.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study showed that the addition of surfactant could indeed improve the ability of \u003cem\u003eB. subtilis\u0026nbsp;\u003c/em\u003eto produce MK-7. Under the conditions of optimizing the surfactant, it was found that the addition of 0.7% Tween-80 could significantly increase the yield of MK-7 and the total synthesis of MK-7 by 80.3%. In addition, according to the measurement of cell membrane permeability, cell morphology, transcription levels of intracellular NADH, NAD\u003csup\u003e+\u003c/sup\u003e and key genes in the MK-7 synthesis pathway, the increase in MK-7 yield in the presence of 0.7% Tween-80 was attributed to the increase in intracellular NADH level. At the same time, this study proved that the improvement of key gene expression level and the enhancement of cell membrane permeability in MK-7 synthesis pathway are beneficial to the exchange of intracellular substances and the accumulation of extracellular MK-7. These results not only provide insights into the mechanism of increasing the yield of MK-7 in the presence of surfactants, but also provide a feasible way for the efficient production of vitamin K2.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYan Liu\u0026minus; College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China; Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, 241000, Wuhu, China; orcid.org/0000-0003-0454-1181; Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeng-jie Zhou\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eJing Wu\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eLiu-xiu Hu\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China; Anhui Zhang Hengchun Pharmaceutical Co., LTD, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eWen-song Hu\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eJun-bao Huang\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eXi-lin Huang\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eXu-li Gao\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eYa-ni Luo\u0026nbsp;\u0026minus;\u0026nbsp;College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China\u003c/p\u003e\n\u003cp\u003eZheng-lian Xue - College of Biology and Food Engineering, Anhui Polytechnic University, 241000, Wuhu, China; Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, 241000, Wuhu, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThe study was supported by the National Nature Science Foundation of China (No. 31871781，31772081), Science and technology project of Wuhu (2020yf62), the National Undergraduate Innovation and Entrepreneurship Program (No. 201910363042) and Anhui Provincial Undergraduate Innovation and Entrepreneurship Program (No. S202010363255).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mengjie Zhou, Jing Wu, Liuxiu Hu, Wensong Hu, Junbao Huang, Xilin Huang, Xuli Gao, Yani Luo, Zhenglian Xue and Yan Liu. The first draft of the manuscript was \u0026nbsp; written by Mengjie Zhou, Jing Wu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e All authors have approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors have no relevant financial or nonfinancial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerenjian A, Chan NL, Mahanama R, Talbot A, Regtop H, Kavanagh J, Dehghani, F (2013) Effect of biofilm formation by \u003cem\u003eBacillus subtilis natto \u003c/em\u003eon menaquinone-7 biosynthesis. Mol Biotechnol 54(2):371\u0026ndash;378. https://doi.org/10.1007/s12033-012-9576-x\u003c/li\u003e\n\u003cli\u003eBerenjian A, Mahanama R, Kavanagh J, Dehghani F (2015) Vitamin K series: current status and future prospects. 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Biotechnol Bioproc E 21(6):777-786. https://doi.org/10.1007/s12257-016-0202-9\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacillus subtilis, Surfactants, Synthetic pathway, Scanning electron microscope, qRT-PCR","lastPublishedDoi":"10.21203/rs.3.rs-2351994/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2351994/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Menaquinone-7 (MK-7), a highly valuable member of the vitamin K2 series, is an essential nutrient for humans. It plays an important role in the treatment of coagulation, osteoporosis, promotion of liver function recovery and prevention of cardiovascular diseases. In this study, in order to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of metabolic synthesis of MK-7 by mutant strain Bacillus subitilis 168 KO-SinR (BS168 KO-SinR) was analyzed by adding surfactants. The results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm, as indicated by scanning electron microscopy and flow cytometry. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. qRT-PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the results of electron microscope showed that the addition of surfactant changed the permeability of cell membrane. The research results of this paper provide a certain reference value for the industrial development of MK-7 prepared by fermentation.","manuscriptTitle":"Improvement of Vitamin K2 Production by Bacillus subtilis mutant with leakage fermentation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-09 15:30:58","doi":"10.21203/rs.3.rs-2351994/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-12-09T08:34:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-07T12:00:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-12-07T08:20:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2022-12-07T03:12:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bf41fa19-2fb8-4471-88be-2994d4582b0b","owner":[],"postedDate":"December 9th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:40:27+00:00","versionOfRecord":{"articleIdentity":"rs-2351994","link":"https://doi.org/10.1007/s11274-023-03671-8","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2023-06-09 21:06:12","publishedOnDateReadable":"June 9th, 2023"},"versionCreatedAt":"2022-12-09 15:30:58","video":"","vorDoi":"10.1007/s11274-023-03671-8","vorDoiUrl":"https://doi.org/10.1007/s11274-023-03671-8","workflowStages":[]},"version":"v1","identity":"rs-2351994","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2351994","identity":"rs-2351994","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}